Post-translational modification involves the introduction of a novel functional group to a protein side chain resulting in changes in enzyme activity, ligand affinity, protein-protein interactions, and protein stability. While the physical and chemical properties of the twenty standard amino acids have been rigorously examined with respect to structure-function relationships, much less is known about the nature, and functional role, of the modified amino acids. This proposal involves the use of state-of-the-art physical chemistry based models of protein energetics to elucidate the nature of the modified sulfotyrosine and methyl-lysine residues and their effects on protein structure, dynamics and binding specificity. The unique nature of sulfotyrosine (sTyr) hydrogen bonds, as distinct from those formed by phosphotyrosine (pTyr), will be studied using potentials of mean force, generated by molecular mechanics calculations in implicit and explicit solvent, on residue pairs involving Tyr, sTyr, and pTyr, and positively charged residues. Hydrogen bonding will further be studied using molecular dynamics simulation of tripeptides Arg-Gly-xTyr and Lys-Gly-xTyr, with xTyr representing unmodified, sulfated, or phosphorylated tyrosine. Molecular dynamics simulation of experimental complexes of the CCR5 and CXCR4 co-receptors, sulfated and unsulfated, will be used to study the effects of sulfotyrosine on the structure and dynamics of these protein systems. Partial charges for the sTyr and pTyr residues in the above studies will be determined using quantum mechanical calculations on the isolated residues. Interactions involving methyl-lysine residues, and the extent to which the strength of these interactions depends on methylation state (mono, di, or trimethylated), will be studied using 1) potentials of mean force, to study hydrogen bonding between mono and dimethyl-lysine and negatively charged amino acids;2) molecular mechanics/implicit solvent (OPLS-AA/GBSA) calculations, to study the effects of steric hindrance and hydrophobic desolvation on methyl-lysine binding proteins in complex with methyl-lysine residues in different methylation states;3) quantum mechanical calculations on mono, di, tri, or tetramethylammonium interacting with benzene, to study cation- @ interactions between methyl-lysine residues and aromatic residues;and 4) molecular dynamics simulation of the B35P1 binding protein in complex with the methylated p53 tumor suppressor to determine how dimethyl-lysine, as distinct from unmodified or monomethyl-lysine, enhances p53 stability and activity. Undergraduate students will be involved in all aspects of this proposal which will serve as excellent training in the use of state-of-the-art chemical simulation software and its application to problems of biological interest. The results of our studies will be used in the development of accurate models for the simulation of modified residues, and may be used to inform structure based therapeutic efforts which target modified protein systems. PUBLIC HEALTH RELEVANCE: Post-translational modification in proteins involves the addition of a chemical group to a protein after it is synthesized in the cell, and plays a key role in expanding the diversity of protein activities. Tyrosine sulfation and lysine methylation, two post-translational modifications which are highlighted in this proposal, are responsible for biological and pathological processes including entry of HIV-1 into the cell, binding of HIV-1 neutralizing antibodies, regulation of genetic expression, and modulation of the activity of the p53 tumor suppressor. The understanding of how tyrosine sulfation and lysine methylation affect protein structure, dynamics, binding specificity, and ultimately functional outcome, is critical to structural biology and therapeutic efforts which target modified protein systems.